The objective of this proposal is to explore the biochemical mechanisms of proteolysis of the excess amounts of newly synthesized normal globin chains in erythro cells of normal and thalassemic individuals. Blood cells from Beta-thalassemic donors will be pulse-labeled with (3H) leucine and incubated subsequently in a non-radioactive medium for several hours. The pool of excess newly synthesized Alpha chains will be isolated by gel filtration chromatography or cellulose acetate electrophoresis, and their catabolism will be measured by the decrease in acid-precipitable 3H-radioactivity. The energy (ATP) and enzyme (protease) dependency of this catabolism will be determined by observing the effect of addition of selective inhibitors such as 2,4-dinitrophenol or N-ethylmaleimide, respectively. A cell-free system for the proteolysis of substrate 3H-Alpha chains will be established, and the roles of the heat stable polypeptide ubiquitin and soluble and stromal enzyme fractions in the proteolytic pathway will be determined. A systematic survey of the type and chemical specificity of the proteases involved will be made by observing the effect of chemical and polypeptide inhibitors with known specificities. These data will indicate the cellular mechanism of control of Alpha chain proteolysis and may suggest the design of chemical agents to interfere with the natural inhibitors of these proteases. This approach may form the basis for enhancement of Alpha chain proteolysis with the concomitant reduction of the inclusion body formation in erythropoietic cells that contributes to the pathophysiology of Beta-thalassemia. Cultured erythroid cells will be used to compare the properties of proteolysis of the small pool of Alpha chains in normal cells with those of the larger pool of excess Alpha chains in Beta-thalassemic cells. The proteolysis of excess, newly synthesized non-Alpha chains in the blood cells of individuals with Alpha-thalassemic disorders, e.g. HbH disease, will also be studied by these procedures.